The Gram-negative a-proteobacteria of the genus Rickettsia are small (0.3-0.5 x 0.8-1.0 m), obligate intracellular organisms. They are categorized into two major groups, the Spotted Fever Group (SFG) and Typhus Group (TG), which can be distinguished by antigenicity and intracellular actin-based motility. Members of this genus are responsible for severe human diseases and several species including R. conorii and R. rickettsii, have been classified as Category B and C Priority Pathogens by the National Institute of Allergy and Infectious Diseases (NIAID) for their potential use as tools for biologica terrorism. We have recently determined that pathogenic rickettsial species sequenced to date posses a gene (in R. conorii, rc1339) whose product has homology to a family of retropepsin-like aspartate proteases (AP) including the HIV-1 protease. We have shown that a purified soluble domain of the protease termed APRcSD (aspartate protease of R. conorii soluble domain) is auto-activated by an internal cleavage event and like other APs, this processing is dependent on a conserved aspartate residue is the catalytic triad domain. We have also demonstrated that protease activity against substrates is not diminished by canonical aspartyl protease inhibitors such as pepstatin, but is blocked by compounds that are specific for HIV-1/HIV-2 proteases and are used to clinically treat HIV infections. Furthermore, we have shown that RC1339/APRc is expressed at the outer-membrane of R. conorii and R. rickettsii and that it is sufficient to mediate the maturation of a conserved surface cell antigen, Sca5/OmpB, a bona fide virulence determinant for rickettsial species. Interestingly, an avirulent strain of R. ricketsii (strain Iowa) contains a defect in OmpB processing, suggesting that the maturation of OmpB and likely other surface antigens is an important event during the infection process. The experiments outlined in this proposal will address the following avenues of research: i. We will characterize the functional role of RC1339/APRc in vitro, define substrates for this enzyme and determine the relevance of differential rc1339 regulation in virulent and avirulent rickettsial species. ii. We will assess the effect of pharmacological inhibition of RC1339 to the rickettsial life cycle and assess the phenotype of an rc1339 mutant in both in vitro and in vivo models of infection.